DESCRIPTION (Investigator's abstract): Spinocerebellar ataxia type 8 (SCA8) is caused by a CTG expansion in an untranslated, endogenous antisense RNA that overlaps the Kelch-like 1 (KLHL1) gene. The KLHL1 promoter and open-reading frame are conserved in mouse, and a KLHL1.antisense transcript (KLHL1AS) is present in mouse as well. We have performed initial characterization of the KLHL1AS and KLHL1 genes in both man and mouse, but we are at this point left with some fundamental questions regarding these genes: 1) What is the normal function of the evolutionarily conserved KLHL1-antisense RNA?, 2) What role does the KLHL1 protein play in the neurons in which it is expressed?, and 3) How does the CTG expansion affect the KLHL1AS RNA and KLHL1 protein, and can these effects explain the neurodegeneration seen in SCA8 patients? The data we have obtained to date have allowed us to make informed hypotheses concerning these questions and to design some in vivo experimental systems to test, refine, and if necessary reformulate these hypotheses. The transcriptional organization of the KLHL1 mRNA and the KLHL1AS transcript suggests that KLHL1AS is most likely a regulator of KLHL1 expression. Based on the homology of KLHL1 to other neuron-specific kelch-like proteins and on our preliminary characterization of this protein, we speculate that KLHL1 may play a role in organizing the actin fibers that help to generate and maintain axons and dendrites. We cannot predict a priori how the SCA8 CTG expansion may affect the KLHL1/KLHL1AS gene system. Both of these genes, however, are specifically expressed in the cerebellum, and so these transcripts are likely candidates for mediating the pathogenic effect of this expansion either directly or through altered antisense interactions. We will perform multiple, iterative modifications of a BAC clone encoding the human KLHLAS gene and the first two exons of KLHL1 in E. coli using homologous recombination. We will then introduce the modified BAC clones into tissue culture cel Is to directly determine how KLHL1 and KLHL1AS interact and measure what effect these interactions have on KLHL1 expression levels. Replacing the CTG repeat in the last KLHL1AS exon with expanded repeats in these clones will also allow us to test how the SCA8 expansion affects KLHL1AS and KLHL1, and how it alters the interactions between these genes. Although we will continue to characterize the structure, protein interactions and cellular and subcellular localization of the KIHI1 protein, we will directly test the role of this protein in normal neuronal development and function by generating a KLHL1 gene knockout in mouse. We have designed our knockout strategy in a way that will allow us to both 1) study the effects of disrupting the KLHL1 gene in a tissue-specific and temporal-specific manner, and 2) determine the effect that altered levels of KLHL1AS transcription has on KLHL1 expression and function.